We propose to fully develop novel assays that can be used in high throughput molecular screening to identify bioactive small molecules that inhibit regulated vesicle exocytosis. Rationale: Regulated vesicle exocytosis plays an essential role in mediating the secretion of physiological signaling molecules. Many examples of hyper- or hyposecretion underlie pathological conditions for which there are currently no drug therapies. Moreover, there are no well-characterized inhibitors of vesicle exocytosis for use in physiological and cell biological studies. As part of our research effort toward elucidating key mechanisms in the pathways of regulated vesicle exocytosis, we seek to identify bioactive small molecules that target and inhibit essential steps in the multi-step vesicle exocytic pathway. Strategy: We are developing novel cell-based fluorescence assays for regulated vesicle exocytosis that can be implemented in high throughput screens (HTS) of chemical diversity libraries. Because there are both common as well as distinct molecular mechanisms associated with vesicle exocytosis in different secretory cell types (e.g., neuroendocrine versus mast cells), we will utilize assays for two cell types with which bioactive small molecules will be characterized as acting either generally or specifically. Secondary assays utilizing amine &enzyme secretion will be used to validate positive hits from the primary screens. Subsequently, molecular assays that are progressively informative will narrow and define the cellular sites of action of the bioactive molecules validated in the secondary screens. Significance: Currently there are no well-characterized small molecules that either generally or specifically affect regulated vesicle exocytosis. Completion of the aims of this proposal will fill an important gap in discovering and characterizing small molecule reagents for cell biological and mechanistic studies of regulated vesicle exocytosis. PUBLIC HEALTH RELEVANCE: There are many disease conditions that are caused by or associated with excessive secretion of hormones, neurotransmitters or inflammatory mediators. For example, in asthma there is excessive secretion of histamine, an inflammatory mediator, and of mucous, which coats the respiratory lining. The work conducted in this application will discover small organic molecules that can be used to inhibit secretion. These molecules will be valuable for understanding the basic process of secretion and their discovery may lead to the development of drugs that prevent secretion under disease conditions.